In photosystem I (PSI), phylloquinone participates to electron transfer as secondary electron acceptor (A1). The phylloquinone biosynthesis pathway, previously characterized by reverse genetic in Synechocystis sp. PCC 6803, involves 8 enzymatic steps from chorismate [1]. In the green alga Chlamydomonas reinhardtii, characterization of phylloquinone biosynthesis was still partial and only one mutant deficient for MEND was characterized [2]. In the present work, we found MENA-H homologs in C. reinhardtii genomic database. In particular, MENF, MEND, MENC, and MENH catalytic domains are present in a single ORF (named PHYLLO by similarity to gene organisation in Arabidopsis). We then took advantage of the fact that a double reduction of plastoquinone (PQ) in PQH2 occurs in anoxia into the A1 site in the mend mutant, interrupting photosynthetic electron transfer [3], to isolate new phylloquinone-deficient strains. UPLC-MS analysis confirmed the absence of phylloquinone in four news mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Despite this loss, men mutants are still able to grow in low light but are high light-sensitive. In low light, the level of active PSII in men mutants is identical to that of the wild-type, but the level of active PSI is reduced by 30-40% as assayed by spectroscopic measurements. This decrease is more pronounced when cells are exposed to high light intensities during 4 hours. The level of active PSI is ~ 10% of wild-type cells and the electron photosynthetic transfer is reduced accordingly. Reorganization of the photosynthetic apparatus following lack of phylloquinone in men mutants is discussed. [less ▲]

In photosystem I (PSI), phylloquinone participates to electron transfer as secondary electron acceptor (A1). The phylloquinone biosynthesis pathway, previously characterized by reverse genetic in Synechocystis sp. PCC 6803, involves 8 enzymatic steps from chorismate [1]. In the green alga Chlamydomonas reinhardtii, characterization of phylloquinone biosynthesis was still partial and only one mutant deficient for MEND was characterized [2]. In the present work, we found MENA-H homologs in C. reinhardtii genomic database. In particular, MENF, MEND, MENC, and MENH catalytic domains are present in a single ORF (named PHYLLO by similarity to gene organisation in Arabidopsis). We then took advantage of the fact that a double reduction of plastoquinone (PQ) in PQH2 occurs in anoxia into the A1 site in the mend mutant, interrupting photosynthetic electron transfer [3], to isolate new phylloquinone-deficient strains. UPLC-MS analysis confirmed the absence of phylloquinone in four news mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Despite this loss, men mutants are still able to grow in low light but are high light-sensitive. In low light, the level of active PSII in men mutants is identical to that of the wild-type, but the level of active PSI is reduced by 30-40% as assayed by spectroscopic measurements. This decrease is more pronounced when cells are exposed to high light intensities during 4 hours. The level of active PSI is ~ 10% of wild-type cells and the electron photosynthetic transfer is reduced accordingly. Reorganization of the photosynthetic apparatus following lack of phylloquinone in men mutants is discussed. [less ▲]

The glyoxylate cycle is essential for growth on C2 compounds such as acetate. In this investigation, for the first time, we have elucidated the subcellular localization of the enzymes of the glyoxylate ... [more ▼]

The glyoxylate cycle is essential for growth on C2 compounds such as acetate. In this investigation, for the first time, we have elucidated the subcellular localization of the enzymes of the glyoxylate cycle in the green microalga Chlamydomonas reinhardtii. Acetyl-CoA synthase and malate dehydrogenase exist as multiple isoforms in this microalga, therefore, we first identified those implicated in the glyoxylate cycle based on the observation that lack of isocitrate lyase (ICL) in a previously identified icl deficient mutantwas correlatedwith specific loss of the other enzymes of the glyoxylate cycle. In this work, we determined that five of the six enzymes associated with the glyoxylate cycle were found to be within peroxisomal microbodies. Citrate synthase, aconitase, malate synthase, malate dehydrogenase, and acetyl-CoA synthase are located in peroxisomal microbodies while isocitrate lyase is cytosolic. Our findings implicate a key role for these cellular compartments in acetate assimilation for Chlamydomonas.Microbodies have only recently been discovered in C. reinhardtii and their existence had been previously debated. The isoform specific subcellular localization determined here suggests that peroxisomal microbodies should be considered in the design of metabolic models for carbon assimilation in C. reinhardtii. [less ▲]

Mitochondrial F1FO-ATP synthase of chlorophycean algae is dimeric. It contains eight orthodox subunits (alpha, beta, gamma, delta, epsilon, OSCP, a and c) and nine atypical subunits (Asa1 to 9). These subunits build the peripheral stalk of the enzyme and stabilize its dimeric structure. The location of the 66.1kDa subunit Asa1 has been debated. On one hand, it was found in a transient subcomplex that contained membrane-bound subunits Asa1/Asa3/Asa5/Asa8/a (Atp6)/c (Atp9). On the other hand, Asa1 was proposed to form the bulky structure of the peripheral stalk that contacts the OSCP subunit in the F1 sector. Here, we overexpressed and purified the recombinant proteins Asa1 and OSCP and explored their interactions in vitro, using immunochemical techniques and affinity chromatography. Asa1 and OSCP interact strongly, and the carboxy-terminal half of OSCP seems to be instrumental for this association. In addition, the algal ATP synthase was partially dissociated at relatively high detergent concentrations, and an Asa1/Asa3/Asa5/Asa8/a/c10 subcomplex was identified. Furthermore, Far-Western analysis suggests an Asa1-Asa8 interaction. Based on these results, a model is proposed in which Asa1 spans the whole peripheral arm of the enzyme, from a region close to the matrix-exposed side of the mitochondrial inner membrane to the F1 region where OSCP is located. 3D models show elongated, helix-rich structures for chlorophycean Asa1 subunits. Asa1 subunit probably plays a scaffolding role in the peripheral stalk analogous to the one of subunit b in orthodox mitochondrial enzymes. [less ▲]

Microalgae are a source of numerous compounds that can be used in many branches of industry. Synthesis of such compounds in microalgal cells can be amplified under stress conditions. Exposure to various ... [more ▼]

Microalgae are a source of numerous compounds that can be used in many branches of industry. Synthesis of such compounds in microalgal cells can be amplified under stress conditions. Exposure to various metals can be one of methods applied to induce cell stress and synthesis of target products in microalgae cultures. In this review, the potential of producing diverse biocompounds (pigments, lipids, exopolymers, peptides, phytohormones, arsenoorganics, nanoparticles) from microalgae cultures upon exposure to various metals, is evaluated. Additionally, different methods to alter microalgae response towards metals and metal stress are described. Finally, possibilities to sustain high growth rates and productivity of microalgal cultures in the presence of metals are discussed. [less ▲]

In this work, the effect of beech (Fagus sylvatica) wood acid hydrolysate on growth of Chlorella sorokin-iana was evaluated. Experiments carried out in this study show that neutralized wood acid ... [more ▼]

In this work, the effect of beech (Fagus sylvatica) wood acid hydrolysate on growth of Chlorella sorokin-iana was evaluated. Experiments carried out in this study show that neutralized wood acid hydrolysate can vastly improve Chlorella growth, due to the presence of organic carbon. However, simultaneously the suppression of Chlorella growth at the onset of cultivation was observed, presumably due to inhibitory substances, and this effect was more pronounced with the increase of hydrolysate dosage. Beech wood acid hydrolysate can be a valuable feedstock to stimulate Chlorella growth, on condition that inhibitory level of hydrolysate loading is avoided. [less ▲]

The unicellular green alga Chlamydomonas reinhardtii is a model organism for studying energetic metabolism. Most mitochondrial respiratory-deficient mutants characterized to date have been isolated on the ... [more ▼]

The unicellular green alga Chlamydomonas reinhardtii is a model organism for studying energetic metabolism. Most mitochondrial respiratory-deficient mutants characterized to date have been isolated on the basis of their reduced ability to grow in heterotrophic conditions. Mitochondrial deficiencies are usually partly compensated by adjustment of photosynthetic activity and more particularly by transition to state 2. In this work, we explored the opportunity to select mutants impaired in respiration and/or altered in dark metabolism by measuring maximum photosynthetic efficiency by chlorophyll fluorescence analyses (FV/FM). Out of about 2900 hygromycin-resistant insertional mutants generated from wild type or from a mutant strain deficient in state transitions (stt7 strain), 22 were found to grow slowly in heterotrophic conditions and 8 of them also showed a lower FV/FM value. Several disrupted coding sequences were identified, including genes coding for three different subunits of respiratory-chain complex I (NUO9, NUOA9, NUOP4) or for isocitrate lyase (ICL1). Overall, the comparison of respiratory mutants obtained in wild-type or stt7 genetic backgrounds indicated that the FV/FM value can be used to isolate mutants severely impaired in dark metabolism. [less ▲]

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential ... [more ▼]

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa. [less ▲]

Chlorophyll is a photosynthetic pigment available abundantly in microalgae and terrestrial plants. This pigment found applications in pharmaceutical, cosmetic and food products as a wound healing ... [more ▼]

Chlorophyll is a photosynthetic pigment available abundantly in microalgae and terrestrial plants. This pigment found applications in pharmaceutical, cosmetic and food products as a wound healing, antioxidant or coloring agent. Recent reports suggest that chlorophyll can also be used as a biomordant to enhance the dyeing process of textile products, but also as a textile dye with antimicrobial properties. In this presentation, different aspects of chlorophyll production are discussed. Firstly, numerous plant biomass types as potential sources of chlorophyll are presented. Subsequently, different methods for chlorophyll extraction and separation from plant biomass are described. Finally, possibilities of chlorophyll implementation into textile products on industrial scale are evaluated. [less ▲]

Euglena gracilis is a secondary green alga related to trypanosomes that derives from a secondary endosymbiosis between a phagotrophic ancestor and a prasinophycean green alga. Our general objective is to ... [more ▼]

Euglena gracilis is a secondary green alga related to trypanosomes that derives from a secondary endosymbiosis between a phagotrophic ancestor and a prasinophycean green alga. Our general objective is to study the metabolic interactions established between the secondary plastid and the mitochondrion after the endosymbiotic event and to determine the phylogenetic origin of the genes encoding the proteins involved in the energetic pathways. As a first step, we analysed the subunit composition of the mitochondrial respiratory chain, both in silico and by targeted proteomics, to assess the extent of its similitude with the respiratory chain of Trypanosomatidae. We have shown that Euglena shares many additional subunits with trypanosomes, which suggests that these subunits are not especially associated to a parasitic lifestyle. As a second step, we sequenced the total transcriptome of Euglena and determined the phylogenetic origin of each predicted transcript using a database of about 1000 complete proteomes representing the diversity of life. These analyses confirmed that Euglena recruited its genes from a very diverse set of sources. As a third step, we performed a high-throughput analysis of the mitochondrial proteome of Euglena. Our MS/MS experiments, taking advantage of the availability of our transcriptome, mostly recovered mitochondrial proteins, which indicates that our mitochondrial extracts were quite pure. The identified proteins encompassed about 15 different mitochondrial pathways. We are now in the process of comparing the expression levels of both the transcripts and the corresponding proteins across a range of culture conditions selected to differently stimulate the mitochondrion and the plastid. [less ▲]